Cyclosporins

ABSTRACT

Nonimmunosuppressant cyclosporin derivatives having cyclophilin-binding activity, for example, the compound, [MeIle]4-ciclosporin, are useful in inhibiting HIV-1 replication in treating AIDS and AIDS related disorders.

This is a continuation of application Ser. No. 08/232,795, filed Apr.25, 1994, which in turn is a continuation of application Ser. No.08/057,067, filed May 3, 1993, which in turn is a continuation ofapplication Ser. No. 07/785,959, filed Oct. 31, 1991, all of which arenow abandoned.

The present invention relates to novel cyclosporins, their use aspharmaceuticals and pharmaceutical compositions comprising them, as wellas to processes for their production.

The cyclosporins comprise a class of structurally distinctive, cyclic,poly-N-methylated undecapeptides, commonly possessing pharmacological,in particular immunosuppressive, anti-inflammatory and/or antiparasiticactivity. The first of the cyclosporins to be isolated was the naturallyoccurring fungal metabolite Ciclosporin or Cyclosporine, also known ascyclosporin A and commercially available under the Registered TrademarkSANDIMMUN® or SANDIMMUNE®. Ciclosporin is the cyclosporin of formula A.##STR1## where MeBmt represents theN-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)threonyl residue of formula B##STR2## in which -x-y- is --CH═CH-- (trans).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the IR spectrum in CH₂ Cl₂ of ciclosporin.

FIG. 2 is the proton NMR spectrum in CDCl₃ of ciclosporin.

FIG. 3 is a 300 MHz HNMR spectrum in CDCl₃ of the product of Example 5.

Since the original discovery of Ciclosporin, a wide variety of naturallyoccurring cyclosporins have been isolated and identified and manyfurther non-natural cyclosporins have been prepared by total- orsemi-synthetic means or by the application of modified culturetechniques. The class comprised by the cyclosporins is thus nowsubstantial and includes, for example, the naturally occurringcyclosporins A through Z cf. Traber et al; 1, Helv. Chim. Acta, 60,1247-1255 (1977); Traber et al; 2, Helv. Chim. Acta, 65, 1655-1667(1982); Kobel et al, Europ. J. Applied Microbiology and Biotechnology,14, 273-240 1982); and von Wartburg et al, Progress in Allergy, 38,28-45, 1986)!, as well as various non-natural cyclosporin derivativesand artificial or synthetic cyclosporins including dihydro-cyclosporinsin which the moiety -x-y- of the MeBmt residue (formula B above) issaturated to give -x-y-═--CH₂ --CH₂ --!; derivatised cyclosporins (e.g.in which the 3'--O-atom of the MeBmt residue is acylated or a furthersubstituent is introduced at the α-carbon atom of the sarcosyl residueat the 3-position); cyclosporins in which the MeBmt residue is presentin isomeric form (e.g. in which the configuration across positions 6'and 7' of the MeBmt residue is cis rather than trans); and cyclosporinsin which variant amino acids are incorporated at specific positionswithin the peptide sequence, e.g. employing the total synthetic methodfor the production of cyclosporins developed by R. Wenger--see e.g.Traber et al. 1, Traber et al, 2 and Kobel et al., loc. cit.; U.S. Pat.Nos. 4,108,985, 4,220,641, 4,288,431, 4,554,351, 4,396,542 and 4,798,823European Patent Publications Nos. 34,567A, 56,782A, 300,784A, 300,785Aand 414,632A; International Patent Publication No WO 86/02080 and UKPatent Publications Nos. 2,206,119 and 2,207,678; Wenger 1, Transpl.Proc., 15 Suppl. 1:2230 (1983); Wenger 2., Angew. Chem. Int. Ed. 24 77(1985) and Wenger 3., Progress in the Chemistry of Organic NaturalProducts, 50, 123 (1986).

The class comprised by the cyclosporins is thus now very large indeedand includes, for example, Thr!² -, Val!² -, Nva!² - and Nva!² - Nva!⁵-Ciclosporin (also known as cyclosporins C, D, G and M respectively),3-O-acetyl-MeBmt!¹ -Ciclosporin (also known as cyclosporin A acetate),Dihydro-MeBmt!¹ - Val!2-Ciclosporin (also known as dihydro-cyclosporinD), (D)Ser!⁸ -Ciclosporin, MeIle!¹¹ -Ciclosporin, (D)MeVal!¹¹-Ciclosporin (also known as cyclosporin H), MeAla!⁶ -Ciclosporin,(D)Pro!³ -Ciclosporin and so on.

In accordance with conventional nomenclature for cyclosporins, these aredefined throughout the present specification and claims by reference tothe structure of Ciclosporin (i.e. Cyclosporin A). This is done by firstindicating those residues in the molecule which differ from thosepresent in Ciclosporin and then applying the term "Ciclosporin" tocharacterise the remaining residues which are identical to those presentin Ciclosporin. At the same time the prefix "dihydro" is employed todesignate cyclosporins wherein the MeBmt residue is hydrogenated(dihydro-MeBmt) i.e. where -x-y- in formula B is --CH₂ --CH₂ --. ThusThr!² -Ciclosporine is the cyclosporin having the sequence shown inFormula A but in which αAbu at the 2-position is replaced by Thr, andDihydro-MeBmt!¹ - Val!² -Ciclosporin is the cyclosporin having thesequence shown in Formula A but in which the MeBmt residue at position 1is hydrogenated and αAbu at the 2-position is replaced by Val.

In addition, amino acid residues referred to by abbreviation, e.g. Ala,MeVal, αAbu etc. are, in accordance with conventional practice, to beunderstood as having the (L)-configuration unless otherwise indicated,e.g. as in the case of "(D)Ala". Residue abbreviations preceded by "Me"as in the case of "MeLeu", represent α-N-methylated residues. Individualresidues of the cyclosporin molecule are numbered, as in the art,clockwise and starting with the residue MeBmt or dihydro-MeBmt inposition 1. The same numerical sequence is employed throughout thepresent specification and claims.

It is now well established that Ciclosporin acts by interfering with theprocess of T cell activation by blocking transcription initiation ofIL-2, although the precise mechanism has not yet been elucidated.Ciclosporin has been shown to form a complex with a 17 kD cytosolicprotein (cyclophilin) that occurs in many cell types and has been shownto be identical to peptidyl-prolyl cis-trans isomerase, an enzymeinvolved in protein folding. Up to now, however, it has not been clearwhether binding to cyclophilin is directly correlated withimmunosuppressive activity in cyclosporins, or indeed whethercyclophilin binding is itself a sufficient criterion forimmunosuppressive activity.

It has now been found that there are cyclosporins which bind strongly tocyclophilin, but are not at all immunosuppressive. It therefore followsthat binding to cyclophilin is a necessary, but not a sufficient,criterion for immunosuppressant activity.

The present invention provides cyclosporins which are active againstHIV-1 replication.

Human immune deficiency virus (HIV) infects preferentially T-helper (T4)lymphocytes, although it replicates also in various other cell types,especially those of the monocytic lineage. It causes a slowlyprogressing disease of the immune system characterised by a gradualT4-cell destruction, named AIDS. Other immunological abnormalities ofAIDS are increase of cytotoxic/suppressor (T8) lymphocytes, a defect inthe antigen presentation/recognition process and polyclonal activationof B-cells. The mechanism of T4-cell destruction is still not clear.Relatively few T4-cells seem to be infected, thus, a direct cytopathiceffect caused by the virus may not be the only reason for T4-celldepletion. It has been hypothesised that T4-cell destruction could beamplified by an autoimmune process triggered by HIV-producing orHIV-protein-coated T4-cells. This continuous antigenic stimulation maylead to a state of permanent activation of T4-cells which would enhanceHIV-replication in these cells and expand T-cytotoxic clones. UninfectedT4-cells may be rendered antigenic by binding exogenous viral gp120 totheir CD4 molecules and would thus be a target of a T-cytotoxicresponse.

U.S. Pat. No. 4,814,323 discloses that Ciclosporin has activity againstAIDS, and that in general "cyclosporins known as immunosuppressors" maybe useful in this indication. There is no suggestion thatnon-immunosuppressive cyclosporins might be expected to have thisproperty.

Surprisingly, it has now been found that cyclosporins which bind tocyclophilin, but are not immunosuppressive, exhibit an inhibitory effectupon HIV-1 replication.

A cyclosporin is considered as binding to cyclophilin if it binds tohuman recombinant cyclophilin at least one fifth as well as doesCiclosporin in the competitive ELISA test described by Quesniaux in Eur.J. Immunol. 1987 17 1359-1365. In this test, the cyclosporin to betested is added during the incubation of cyclophilin with coatedBSA-Ciclosporin and the concentration required to give a 50% inhibitionof the control reaction without competitor is calculated (IC₅₀). Theresults are expressed as the Binding Ratio (BR), which is the log to thebase 10 of the ratio of the IC₅₀ of the test compound and the IC₅₀ in asimultaneous test of Ciclosporin itself. Thus a BR of 1.0 indicates thatthe test compound binds cyclophilin one factor of ten less well thandoes Ciclosporin, and a negative value indicates binding stronger thanthat of Ciclosporin.

The cyclosporins active against HIV have a BR lower than 0.7, (sincelog₁₀ 5=0.7 approx), preferably equal to or lower than zero.

A cyclosporin is considered to be non-immunosuppressive when it has anactivity in the Mixed Lymphocyte Reaction (MLR) of no more than 5%,preferably no more than 2%, that of Ciclosporin. The Mixed LymphocyteReaction is described by T.Meo in "Immunological Methods", L. Lefkovitsand B. Peris, Eds., Academic Press, N.Y. pp. 227-239 (1979). Spleencells (0.5×10⁶) from Balb/c mice (female, 8-10 weeks) are co-incubatedfor 5 days with 0.5×10⁶ irradiated (2000 rads) or mitomycin C treatedspleen cells from CBA mice (female, 8-10 weeks). The irradiatedallogeneic cells induce a proliferative response in the Balb c spleencells which can be measured by labeled precursor incorporation into theDNA. Since the stimulator cells are irradiated (or mitomycin C treated)they do not respond to the Balb/c cells with proliferation but do retaintheir antigenicity. The IC₅₀ found for the test compound in the MLR iscompared with that found for Ciclosporin in a parallel experiment.

The activity of cyclosporins as inhibitors of HIV-1 replication may bedemonstrated in the following test systems:

1. Inhibition of HIV-1-induced cytopathic effect in MT4-cells

The assay procedure described by Pauwels et al., J. Virol. Meth. 20/309(1988), is used with minor modifications. The HTLV-I-transformed T4-cellline, MT4, which was previously shown to be highly permissive forHIV-infection, is used as the target cell. Inhibition of HIV-1, strainHTLV-IIIB-induced cytopathic effect is determined by measuring theviability of both HIV-infected and mock-infected cells. Viability isassessed spectrophotometrically via in situ reduction of3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT).Virus-infected and uninfected cultures without compound are included ascontrols as are uninfected cells treated with compound. The cellconcentration is chosen so that the number of cells per ml increases bya factor of 10 during the 4 days of incubation in the mock-infectedcultures. Virus inoculum is adjusted so as to cause cell death in 90% ofthe target cells after 4 days of incubation. The virus is adsorbed ontoa 10-fold concentrated cell suspension at 37° C. for 1 hour. Then, theinfected cells are diluted 1:10 and added to microtiter platescontaining the test compound.

2. Cytotoxicity

To evaluate the anti-HIV potential in additional cell lines andparticularly in a monocytic cell line (U937), cellular toxicity of thetest compound to these cell lines is first assessed. Jurkat and U937cell suspensions are adjusted to 1×10⁵ cells/ml and incubated in thepresence of various concentrations of test compound. After 48 hours theamounts of cells per ml are compared by staining with MTT. Cytoxicity inMT4 cells may be measured in the same way.

3. Inhibition of HIV-1 replication in Jurkat and U937 cells

The T4-cell line Jurkat and the monocytic cell line U937 are infected bysuspending the cells 10-fold concentrated in virus solution. Adsorptionis allowed for 2 hours at 37° C. The cells are then spun down, theinoculum is removed and the cells are resuspended at their originalconcentration in fresh culture medium containing test compound. Thus,the substance is added post adsorption. At days 2, 5, 8, 12, 15 and 19post-infection, aliquots of the infected cultures are removed. Cells arespun down and the supernatants collected. The concentration of viral p24antigen is determined in the supernatants by means of a commercial ELISAkit and serves as a parameter for virus production. After each removalof aliquots, cells are counted and adjusted to 2×10⁵ cells/ml by addingfresh medium containing the test compound at the particularconcentration.

Of the compounds according to the invention, i.e. non-immunosuppressive,cyclophilin-binding cyclosporins active against HIV-1 (ActiveCompounds), some are novel and others are known; however the anti-HIVactivity of the known Active Compounds has not previously beendisclosed, and in many cases the known Active Compounds have not beendisclosed to have any pharmaceutical activity whatsoever.

It is found that many of the Active Compounds have structures differingfrom that of Ciclosporin specifically at the 4 and/or 5 positions.

One group of Active Compounds are cyclosporins in which the MeLeu groupat position 4 is replaced by a different N-methylated amino acid, forexample γ-hydroxy-MeLeu, MeIle, MeVal, MeThr or MeAla. In addition toMeIle and MeThr, the allo-forms MeaIle and MeaThr may also be used. Inthe allo-form, the stereochemistry at the β-position has the oppositeconfiguration to that of the natural amino acid, so that the normal formand the allo-form constitute a pair of diastereoisomers.

A further group of Active Compounds is that in which Val at the5-position is replaced by an N-alkyl-, preferably N-methyl-, amino acid.Preferably the amino acid which is N-alkylated is Val or Leu. Preferablythe hydrogen of the imino group of Val!⁵ is replaced by a non-branchedC₁₋₆ alkyl group, preferably methyl, ethyl or n-propyl, particularlymethyl. The latter preferred group of Active Compounds are all novel.

Additionally or alternatively, certain Active Compounds may differ fromCiclosporin at the 1, 2, 3, and/or 6 positions. A preferred group ofActive Compounds is constituted by the compounds of Formula I: ##STR3##in which W is MeBmt, dihydro-MeBmt or 8'-hydroxy-MeBmt;

X is αAbu, Val, Thr, Nva or 0-methyl threonine (MeOThr);

R is Sar or (D)-MeAla;

Y is MeLeu, γ-hydroxy-MeLeu, MeIle, MeVal, MeThr, MeAla, MeaIle orMeaThr;

Z is Val, Leu, MeVal or MeLeu; and

Q is MeLeu, γ-hydroxy-MeLeu or MeAla;

provided that when Y is MeLeu then either Z is MeVal or MeLeu, or W is8'-hydroxy-MeBmt.

The groups W,X,Y,Z,Q and R have, independently, the following preferredsignificances:

W is preferably W' where W' is MeBmt or dihydro-MeBmt;

X is preferably X' where X' is αAbu or Nva, more preferably X" where X"is αAbu;

R is preferably R' where R' is Sar;

Y is preferably Y' where Y' is γ-hydroxy-MeLeu, MeVal, MeThr or MeIle;

Z is preferably Z' where Z' is Val or MeVal; and

Q is preferably Q' where Q' is MeLeu;

One especially preferred group of Active Compounds are the compounds ofFormula I in which W is W', X is X', Y is Y', Z is Z', Q is Q' and R isR'.

Particularly preferred compounds of Formula I are:

a) dihydro-MeBmt!¹ - γ-hydroxy-MeLeu!⁴ -Ciclosporin

b) MeVal!⁴ -Ciclosporin

c) MeIle!⁴ -Ciclosporin

d) MeThr!⁴ -Ciclosporin

e) γ-hydroxy-MeLeu!⁴ -Ciclosporin

f) Nva!² - γ-hydroxy-MeLeu!⁴ -Ciclosporin

g) γ-hydroxy-MeLeu!⁴ - γ-hydroxy-MeLeu!⁶ -Ciclosporin

h) MeVal!⁵ -Ciclosporin

i) MeOThr!² - (D)MeAla!³ - MeVal!⁵ -Ciclosporin

j) 8'-hydroxy-MeBmt!¹ -Ciclosporin

In addition, certain compounds not within the scope of Formula I areActive Compounds, for example k) MeAla!⁶ -Ciclosporin, and 1)γ-hydroxy-MeLeu!⁹ -Ciclosporin.

The invention also provides novel Active Compounds, which are compoundsof Formula II: ##STR4## where W', X, R, Y and Z are as defined above,provided that 1) when Y is MeLeu or MeAla then Z is MeVal or MeLeu and2) when W' is MeBmt, R is Sar, and Y is γ-hydroxy-MeLeu, then Z is otherthan Val.

A preferred group of novel Active Compounds consists of the compounds ofFormula II in which X is X", Y is Y' and Z is Z', provided that when W'is MeBmt, Y' is other than γ-hydroxy-MeLeu.

Particularly preferred novel Active Compounds are the compounds a), b),c), d), f) and h) above. Certain of these compounds, for examplecompounds b) and c), are found to block the immunosuppressive action ofCiclosporin by blocking its binding to cyclophilin, and thus act asCiclosporin antagonists.

The Active Compounds may be obtained in a variety of ways, which may beclassified as:

1) Fermentation

2) Biotransformation

3) Derivatisation

4) Partial Synthesis

5) Total Synthesis.

1) Certain of the Active Compounds are produced as by-products of thefermentation of original or modified strains of Ciclosporin-producingorganisms such as Tolypocladium inflatum Gams, as exemplified by theproduction of compound c), described in Example 1 below.

2) Other Active Compounds, including the known compounds j) and 1) aremetabolites of Ciclosporin, and can be isolated by chromatographicmethods from the urine of humans or animals dosed with Ciclosporin.Furthermore, these and other metabolic transformations are possibleusing microorganisms, for example the production of compounds e) and g)by biotransformation of cyclosporin A, as described in Examples 2 and 3,or that of compound f) by biotransformation of cyclosporin G (Example4). These examples demonstrate a novel process for the preparation ofcyclosporins having one or more γ-hydroxy-MeLeu residues, comprising thesteps of culturing a novel modified strain of Sebekia benihana, adding acyclosporin having one or more MeLeu residues, and isolating the productfrom the fermentation broth.

3) By derivatisation is meant that natural or synthetic cyclosporins maybe converted into Active Compounds by one or more chemical reactions inwhich one or more of the amino acids are modified without the peptidebonds being opened and reformed. For example, the class of ActiveCompounds in which the Val at position 5 is N-alkylated may be obtainedby reacting the corresponding cyclosporin having Val at position 5 withbutyllithium, followed by reaction with an alkylating agent, asexemplified for compound h) in Example 5 and for compound i) in Example6.

As further examples, compound a) may be prepared by hydrogenation ofcompound e) (Example 7), and compound j), which is also a majormetabolite of Ciclosporin, may be prepared from the known compoundCiclosporin acetate as described in Example 8.

4) The term partial synthesis is used to mean a series of chemicalreactions in which the ring of a natural cyclosporin is opened, one ormore amino acids are removed, different amino acids are added, and thering is closed again.

5) The total synthesis of cyclosporins may be carried out by building upa linear undecapeptide and cyclising, as described by Wenger (loc.cit.), see also U.S. Pat. Nos. 4,396,542 and 4,798,823. In principle,any cyclosporin may be prepared by the total synthesis route, althoughwhere one of the other methods is available, this may well be moreconvenient than total synthesis. Total synthesis may be used for thepreparation of compound d) (Example 9), and for the known metabolitecompound 1).

Compound k) is a known substance whose properties have been described byQuesniaux et al (Mol. Immunol. 24 1159 1987) and which can also beprepared by total synthesis. For example, a total synthesis of thiscompound is described in U.S. Pat. No. 4,914,188.

EXAMPLE 1

MeIle!⁴ -Ciclosporin (Compound c)

Producing Strain

Compound c) is obtained by fermentation of the fungal strainTolypocladium inflatum Cy E 4556, deposited at the Deutsche Sammlung furMikroorganismen under the provisions of the Budapest treaty on 24 Jul.1991 under the accession number DSM 6627. This strain is a mutant of thestrain NRRL 8044 of the species Tolypocladium inflatum Gams, and istaxonomically identical with the parent strain, which has been fullydescribed, e.g. in British Patent 1,491,509.

Culture 1. Agar starting culture: Agar slant cultures of the strain DSM6627 are grown for 14 days at 27° C. on the following agar medium:

    ______________________________________                                        Yeast extract (Gistex) 4      g                                               Malt extract (Wander)  20     g                                               Agar                   20     g                                               Demineralised water to 1000   ml.                                             ______________________________________                                    

The medium has a pH of 5.4-5.6 and is sterilised for 20 min. at 120° C.

2. Preculture: Spores from the mycelium of 4 starting cultures aresuspended in 40 ml of a 0.9% salt solution. A series of 500 mlErlenmeyer flasks each containing 100 ml of preculture medium areinoculated each with 20 ml of this suspension. The composition of thepreculture medium is as follows:

    ______________________________________                                        Casein (Amber EHC)     25     g                                               Maltose                75     g                                               KH2PO4                 1      g                                               KCl                    2.5    g                                               Demineralised water to 1000   ml                                              ______________________________________                                    

The medium is adjusted to pH 5.2-5.5 with HCl, then sterilised for 20min. at 120° C. The precultures are fermented for 24 hr. at 27° C. on arotary shaker at 200 rpm with an eccentricity of 50 mm.

3. Intermediate culture: One 25-liter steel fermenter containing 20liters of preculture medium is inoculated with 200 ml of preculture. Theintermediate culture is fermented during 5 days at 27° C., with astirring rate of 150 rpm and a rate of air flow of 0.5 l/min. per litermedium at 0.5 bar pressure.

4. Main culture: 100 liter of preculture medium are inoculated with 10 lof intermediate culture and fermented in a 120-liter steel fermenter. Tothis medium is added 4 g/l D-Threonin, sterilised by filtration.Fermentation is carried out at 27° C. for 14 days, with a stirring rateof 70 rpm and a rate of air flow of 0.4 l/min per liter medium at 0.5bar pressure being used for the 5 first days, the stirring rate thenbeing increased to 100 rpm and the air flow rate to 0.5 l/min for theremainder of the fermentation period.

Isolation: The mycelium is separated from the culture medium andextracted in a Turrax apparatus by crushing and stirring 3 times with 10liters of methanol/water (9:1 by vol.). The crushed mycelium isseparated from the solvent by suction-filtration and the combinedfiltrates are concentrated by evaporation under vacuum at a temperatureof 40° C. until the vapour consists mainly of water alone. The obtainedmixture is extracted four times using 2 liters 1,2-dichloroethane ateach extraction and the combined 1,2-dichloroethane solutions areconcentrated by evaporation under vacuum at a temperature of ₄₀ ° C.

The residue is subjected to silica gel column chromatography (10 kg ofsilica gel, granulate size 0.02-0.045 mm, "Grace") using ethylacetate/water as eluent (fractions of 2.5 l). Fractions 20-23,containing MeIle!⁴ -Ciclosporin are pooled and then further separated bysilica gel column chromatography (600 g of silica gel, granulate size0.04-0.063 mm, "Merck") using chloroform/methanol (98:2 by vol.) aseluent (fraction size 300 ml). Further purification is achieved bysilica gel column chromatography (400 g of silica gel, granulate size0.04-0.063 mm, "Merck") using methylene chloride/methanol (98:2 by vol.)as eluent (fraction size 200 ml) yielding pure MeIle!⁴ -Ciclosporin asan amorphous white powder: m.p. 155°-158° C.; α!20/D=-235° (c=0.68 inCHCl₃) and -193° (c=0.74 in CH₃ OH).

The IR spectrum in CH₂ Cl₂ is as shown in FIG. 1, and the proton NMRspectrum in CDCl₃ is as shown in FIG. 2.

EXAMPLE 2

γ-hydroxy-MeLeu!⁴ -Ciclosporin (Compound e)

Compound e) is obtained from biotransformation of Ciclosporin by themicroorganism Sebekia benihana. The original strain used is named NRLL11111 and belongs to the species Sebekia benihana (Dietz and Li:Sebekia, a new genus of the family Actinoplanaceae. Abstrs. 82ndAnn.Meet.Amer.Soc.Microbiol., 163, Atlanta, 1982). This strain is ableto hydroxylate novobiocin. The subcultured strain used for thepreparation of compound e and related compounds has been deposited atthe German Collection of Microrganisms (D-3300 Braunschweig) under thenumber DSM 6182.

1. Agar starting culture: Agar slant cultures of the strain DSM 6182 aregrown for 10 days at 27° C. on the following agar medium:

    ______________________________________                                        Glucose                   10    g                                             Starch (soluble)          20    g                                             Yeast extract (Gistex)    5     g                                             Peptone (N-Z-Amine Typ A, Sheffield)                                                                    5     g                                             CaCO.sub.3                1     g                                             Agar (Bacto)              18    g                                             Demineralised water to up to 1 Liter.                                         ______________________________________                                    

pH: neutralised at 7 with NaOH/H₂ SO₄

Sterilisation: 120° C./20 min.

2. Preculture: Spores and mycelium of one starting culture are suspendedin 10 ml of a 0.9% salt solution. A series of 200 ml Erlenmyer flaskseach containing 50 ml of preculture medium are inoculated each with 5 mlof this suspension. The composition of the preculture medium is asfollows:

    ______________________________________                                        Glucose                   7     g                                             Starch (soluble)          10    g                                             Yeast extract (Gistex)    4.5   g                                             Malt extract (liquid, Wander)                                                                           10    g                                             Peptone (N-Z-Amine Typ A, Sheffield)                                                                    2.5   g                                             CaCO.sub.3                1     g                                             Trace solution Nr 235     1     ml                                            Demineralised water to up to 1 Liter.                                         ______________________________________                                    

pH neutralised to 7 with NaOH/H₂ SO₄

Sterilisation: 120° C./20 min.

Trace solution Nr 235:

    ______________________________________                                        H.sub.3 BO.sub.3        0.1    g                                              FeSO.sub.4.7H.sub.2 O   5      g                                              KI                      0.05   g                                              CoCl.sub.2.6H.sub.2 O   2      g                                              CuSO.sub.4.5H.sub.2 O   0.2    g                                              MnCl.sub.2.4H.sub.2 O   2      g                                              ZnSO.sub.4.7H.sub.2 O   4      g                                              Demineralised water up to 999 ml                                              H.sub.2 SO.sub.4 (97%)  1      ml                                             ______________________________________                                    

The precultures are fermented for 4 days at 27° C. on a rotary shaker at200 rpm with an eccentricity of 50 mm.

3. Intermediate culture: A series of 200 ml Erlenmeyer flasks eachcontaining 50 ml of the preculture medium are inoculated each with 5 mlof preculture. The intermediate culture is fermented for 3 days at 27°C. on a rotary shaker at 200 rpm with an eccentricity of 50 mmm.

4. Main culture: A series of 500 ml Erlenmeyer flasks each containing 50ml of the main medium (total 12 liters) are inoculated each with 5 ml ofintermediate culture. These cultures are fermented for 3 days at 27° C.on a rotary shaker at 200 rpm with an eccentricity of 50 mm. After 24hours cyclosporin A (7.5 mg) solubilized in methanol is added to eachmain culture (=150mg/L).

The composition of the main culture medium is as follows:

    ______________________________________                                        Cerelose                10     g                                              Dextrine                10     g                                              Starch (soluble)        10     g                                              Yeast extract (Gistex)  2.5    g                                              Soyabean flour (Nurupan, Edelsoya)                                                                    12.5   g                                              K.sub.2 HPO.sub.4       0.25   g                                              KH.sub.2 PO.sub.4       0.12   g                                              MgSO.sub.4.7H.sub.2 O   0.10   g                                              CaCl.sub.2.6H.sub.2 O   0.05   g                                              Trace element solution AC-1                                                                           1      ml                                             Demineralised water up to 1 Liter.                                            ______________________________________                                    

pH: adjusted at 7.2-7.5 (KOH/H₂ SO₄)

Sterilisation: 120° C./ 20 min.

Trace element solution AC-1: This solution has the same composition asthe solution Nr 235 with the addition of: (NH₄)₆ Mo₇ O₂₄ 0.2 g.

5. Isolation: The mycelium is separated from the culture medium and theresulting culture filtrate (13 L) is extracted three times with1,2-dichloroethane using 1.5 L at each extraction. The combined1,2-dichloroethane solutions are evaporated under vacuum at atemperature of 40° C. The crude residue is subjected to Sephadex LH-20gel filtration using methanol as eluent. Those fractions containing thecyclosporin compounds (525 mg) are pooled and chromatographed on silicagel (50 g, granulate size 0.04-0.063 mm, `Merck`) usingchloroform/methanol as eluent. Repeated chromatography using the samesystem yields pure γ-hydroxy-MeLeu!⁴ -Ciclosporin as an amorphous whitepowder, m.p. 150°-153°, α!_(D) ²⁰ -225° (c=0.53 in CHCl₃), -171° (c=0.44in CH₃ OH).

EXAMPLE 3

γ-hydroxy-MeLeu!⁴ - γ-hydroxy-MeLeu!⁶ -Ciclosporin (compound g)

The more polar side fractions originating from the purification ofγ-hydroxy-MeLeu!⁴ -Ciclosporin are further separated by repeated silicagel column chromatography (granulate size 0.04-0.063 mm) usingacetone/hexane 2:1 as eluent followed by chromatography on silica gelusing methyl t.butyl ether/methanol/water 90:9:1 as eluent. The firstfractions contain γ-hydroxy-Leu!⁴ -Ciclosporin which is further purifiedby decolorizing with charcoal yielding the pure compound as an amorphouswhite powder, m.p. 162°-164°, α!_(D) ²⁰ -211° (c=0.50 in CHCl₃), -157°(c=0.52 in CH₃ OH).

The later fractions from the above silica gel column chromatographycontain γ-hydroxy-MeLeu!⁴ - γ-hydroxy-MeLeu!⁶ -Ciclosporin and arefurther purified by decolorizing with charcoal yielding the pure titlecompound as an amorphous white powder, m.p. 157°-160°, α!_(D) ²⁰ -217°(c=0.54 in CHCl₃), -176° (c=0.42 in CH₃ OH).

EXAMPLE 4

Nva!² - γ-hydroxy-MeLeu!⁴ -Ciclosporin (compound f)

This compound is prepared by a procedure analogous to the preparation ofcompound e ( γ-hydroxy-MeLeu!⁴ -Ciclosporin), but using cyclosporin G (Nva!2-Ciclosporin) as starting material. After purification by repeatedsilica gel column chromatography using ethyl acetate saturated withwater and acetone/hexane 2:1 as eluent, respectively, the title compoundis obtained as an amorphous white powder, m.p. 138°-141°, α!_(D) ²⁰-213° (c=0.69 in CHCl₃), -168° (c=0.70 in CH₃ OH).

EXAMPLE 5

MeVal!⁵ -ciclosporin (compound h)

Cyclosporin A (0.60 g=0.5 mmol) dissolved in tetrahydrofuran (20 ml) isincubated with 0.63 ml of a 1.6M solution of butyllithium (1.0 mmol) inhexane. The resulting solution is reacted with dimethyl sulphate (0.1ml; 1.5 mmol) at -78° C. The reaction mixture is slowly warmed to roomtemperature and stirred overnight.

Isolation by flash chromatography (SiO₂, 5% methanol/ether), followed byHPLC (reverse phase) gave the title product. The compound ischaracterised by a 300 MHz ¹ H NMR spectrum in CDCl₃ which is shown inFIG. 3.

EXAMPLE 6

MeOThr!² - (D)MeAla!³ - MeVal!⁵ -Ciclosporin (Compound i)

A mixture of 480 ml THF (absolute) and 6.96g (49.2 mMol) diisopropylamine is cooled to -80° C., and 33.5 ml of a 1.33M solution ofbutyllithuim in hexane (=44.5 mMol) is added slowly through a syringe.The mixture is stirred for 30 min. at -80° C., then a solution of 8 g(6.6 mMol) cyclosporin C ( Thr!² -Ciclosporin) in 120 ml absolute THF isadded through a syringe over 2-3 minutes. The clear solution is stirredfor a further hour at -80° C., then 2.06 ml methyl iodide is slowlyadded.

The mixture is allowed to warm to room temperature over 2 hr, then 40 mlwater is added and the solvents are evaporated on a rotary evaporator at30° C./15 mm Hg. The residue is partitioned between water and ether, andthe ether layer is washed four times with semi-saturated brine, driedover MgSO₄ and evaporated, to give a residue of 8.1 g.

The residue is chromatographed on 1200 g Kieselgel with water-saturatedethyl acetate, to give a crude product which after a furtherchromatography on 200 g Kieselgel with 5% MeOH/CH₂ Cl₂ gives the puretitle product, α!_(D) ²⁰ =-195° (C=1.0 in CHCl₃).

EXAMPLE 7

dihydro-MeBmt!- γ-hydroxy-MeLeu!⁴ -Ciclosporin (compound a)

To a suspension of 200 mg pre-hydrogenated 10% palladium/charcoal in 4ml ethanol is added 1.2 g of γ-hydroxy-MeLeu!⁴ -Ciclosporin (compound e)in 10 ml ethanol and hydrogenation is carried out at room temperatureuntil no more hydrogen is taken up. After removal of the catalyst byfiltration the solution is evaporated, yielding the title compound as anamorphous white powder, m.p. 154°-156°, α!_(D) ²⁰ -225° (c=0.87 inCHCl₃), -169° (c=0.70 in CH₃ OH).

EXAMPLE 8

8'-hydroxy-MeBmt!¹ -Ciclosporin (compound j)

1. 0-acetyl-ω-bromo-MeBmt!¹ -Ciclosporin: A mixture of 25.0 g (20 mmol)of 0-acetyl-MeBt!¹ -Ciclosporin (Traber et al, Helv.Chim. Acta 1982, 65,1653), 4.4 g (25 mmol) of N-bromo succinimide and 400 mg ofazobisisobutyronitrile in 250 ml carbon tetrachloride is heated toreflux for 2.5 hrs. The solvent is evaporated and replaced by ether,filtered from solids, washed with water, dried over magnesium sulfateand evaporated to dryness. The residue is chromatographed over silicagel with ethyl ether/ethyl acetate (4:1) to give 10.7 g (40%) ofamorphous product which is crystallized from ether/hexane to yield 8.4 gof pure substance; m.p. 207°-209° C. Later fractions of the chromatogramcontained an additional 11.2 g of product of slightly lesser quality.

2. 0-acetyl-ω-acetoxy-MeBmt!¹ -Ciclosporin: A mixture of 4.31 g (3.3mmol) of the product of step 1 (contaminated with an estimated 15-20% ofstarting material) and 2.1 g (8 mmol) of tetraethylammonium acetatetetrahydrate in 30 ml of methyl ethyl ketone, containing a catalyticamount of sodium iodide, is heated in an oil bath at 105° C. for 3 hrs.and kept at room temperature over the weekend. The solvent is dilutedwith methyl t-butyl ether and washed with water and brine. The organiclayer is dried over MgSO₄ and evaporated to leave 4.0 g of crude productwhich is purified on a RP-18 reverse phase column (240 g) to yield 3.07g of the title product; m.p. 191°-192° C.

3. ω-hydroxy-MeBmt!¹ -Ciclosporin: A solution of 1.72 g (1.3 mmol) ofthe product of step 2 in 75 m of methanol and a solution of 1.2 g ofsodium in 50 ml of methanol are mixed and kept at r.t. for 2.5 hours.Then the solution is acidified with acetic acid. The solvent isevaporated under reduced pressure and the residue is dissolved in methylt-butyl ether, washed sequentially with water, brine, and sodiumbicarbonate solution, dried over MgSO₄ and evaporated. The crude product(1.6 g) is eluted from a RP-18 column (240 g) with methanol/water 75:15to give 1.5 g of pure product. A sample is crystallized fromether/hexane to give a crystalline product of m.p. 181°-183° C.

EXAMPLE 9

MeThr!⁴ -Ciclosporin (compound d)

The total synthesis of Ciclosporin as described in U.S. Pat. Nos.4,396,542 and 4,798,823 is carried out using MeThr in the 4-position inplace of MeLeu. The product has α!_(D) ²⁰ =-249.6° (c=1.0 in CHCl₃).

EXAMPLE 10

MeVal!⁴ -Ciclosporin (compound b)

The total synthesis of Ciclosporin as described in U.S. Pat. Nos.4,396,542 and 4,798,823 is carried out using MeVal in the 4-position inplace of MeLeu. The product has α!_(D) ²⁰ =-226° (c=0.358 in CHCl₃).

EXAMPLE 11

Immunosuppression and cyclophilin-binding of Active Compounds relativeto Ciclosporin

Table I gives examples of (1) the cyclophilin Binding Ratio (BR) ofActive Compounds as measured in an ELISA assay and (2) theimmunosuppressive activity of Active Compounds relative to Ciclosporinas measured in an MLR assay and expressed as a percentage of activityrelative to Ciclosporin (Immunosuppressive Ratio or IR).

Further explanation of the significance of these values and the methodsfor conducting these tests are set forth supra.

                  TABLE I                                                         ______________________________________                                        Compound                BR(log.sub.10)                                                                         IR (%)                                       ______________________________________                                        a)  dihydro-MeBmt!.sup.1 - γ-hydroxy-MeLeu!.sup.4 -                                             0.1      <1                                           Ciclosporin                                                                   b)  MeVal!.sup.4 -Ciclosporin                                                                         0.1      <1                                           c)  MeIle!.sup.4 -Ciclosporin                                                                         -0.2     <1                                           e)  γ-hydroxy-MeLeu!.sup.4 -Ciclosporin                                                         -0.3     <1                                           f)  Nva!.sup.2 - γ-hydroxy-MeLeu!.sup.4 -Ciclosporin                                            0.4      <1                                           h)  MeVal!.sup.5 -Ciclosporin                                                                         0.4      5.3                                          j)  8'-hydroxy-MeBmt!.sup.1 -Ciclosporin                                                              0.35     1.8                                          k)  MeAla!.sup.6 -Ciclosporin                                                                         -0.4     3.2                                          l)  γ-hydroxy-MeLeu!.sup.9 -Ciclosporin                                                         0.15     2.9                                          ______________________________________                                    

EXAMPLE 12

Anti-HIV activity and cytotoxicity of Active Compounds

Examples of the activity of Active Compounds and Ciclosporin asinhibitors of HIV-1 replication in MT4-cells and the cytotoxicity ofActive Compounds and Ciclosporin in MT4-cells are set forth in Table II.The significance of these numbers and the appropriate methods arediscussed supra.

                  TABLE II                                                        ______________________________________                                                              Cytotoxicity                                                                            Anti-HIV                                      Compound              (μg/ml)                                                                              (IC.sub.50)                                   ______________________________________                                        a)  dihydro-MeBmt!.sup.1 - γ-hydroxy-MeLeu!.sup.4 -                                           16.3      0.12                                          Ciclosporin                                                                   b)  MeVal!.sup.4 -Ciclosporin                                                                       5.4       0.064                                         c)  MeIle!.sup.4 -Ciclosporin                                                                       4.5       0.056                                         e)  γ-hydroxy-MeLeu!.sup.4 -Ciclosporin                                                       15.5      0.34                                          f)  Nva!.sup.2 - γ-hydroxy-MeLeu!.sup.4 -Ciclosporin                                          14.0      0.85                                          h)  MeVal!.sup.5 -Ciclosporin                                                                       4.4       0.45                                          i)  MeOThr!.sup.2 - (D)MeAla!.sup.3 - MeVal!.sup.5 -Ciclosporin                                     >10       0.45                                          j)  8'-hydroxy-MeBmt!.sup.1 -Ciclosporin                                                            10.6      0.56                                          k)  MeAla!.sup.6 -Ciclosporin                                                                       5.2       1.34                                          l)  γ-hydroxy-MeLeu!.sup.9 -Ciclosporin                                                       >10       0.31                                          Ciclosporin           8.45      0.53                                          ______________________________________                                    

The Active Compounds are indicated both for the prevention of AIDS inasymptomatic HIV-positive patients and in the treatment of patientssuffering from AIDS. In the patients in which AIDS has appeared theadministration of the Active Compounds reverse the T4-cell depletionassociated with AIDS, induce regression of AIDS-related disorders, suchas Kaposi's sarcoma, and reduce the liklihood of new opportunisticinfections.

Thus the invention provides a process for the treatment and theprevention of the acquired immunodeficiency syndrome and other disordersinduced by the HIV-1 virus in a patient infected with said virus,comprising administering to said patient an effective amount of anActive Compound of the invention.

The Active Compound may be administered by any conventional route, inparticular enterally, e.g. orally, for example in the form of solutionsfor drinking, tablets or capsules or parenterally, for example in theform of injectible solutions or suspensions. By the intravenous route anindicated daily dosage may be from 1 to 20 mg/kg, preferably from 3 to10 mg/kg, and by the oral route from 1 to 50 mg/kg, preferably from 7 to20 mg/kg.

The toxicity of the Active Compounds is believed to be similar to thatof Ciclosporin. As the Active Compounds are not immunosuppressive,certain side effects of Ciclosporin related to immunosuppression areavoided. Other side effects associated with Ciclosporin, however,particularly nephrotoxicity in long term use, may also be associatedwith the Active Compounds.

Preferred galenic formulations for the Active Compounds include thosebased on microemulsions as described in British Patent Application 2 222770A, which include topical as well as oral forms; also oral andinjectable forms obtained from solid solutions comprising a fatty acidsaccharide monoester, e.g. saccharose monolaurate, as described inBritish Patent Application 2 209 671A. Suitable unit dosage forms fororal administration comprise e.g. from 25 to 200 mg Active Compound perdosage.

    ______________________________________                                        Formulation Example A:                                                        Product of Ex. 1       50.0   mg                                              Glycofurol 75          180.0  mg                                              Miglyol 812            90.0   mg                                              Cremophor RH40         180.0  mg                                              alpha-Tocopherol       0.5    mg                                              Formulation Example B:                                                        Product of Ex. 1       100.0  mg                                              Tetraglycol            20.0   mg                                              Captex 800             20.0   mg                                              Nikkol HCO-40          860.0  mg                                              Butylhydroxytoluene(BHT)                                                                             1.0    mg                                              Formulation Example C:                                                        Product of Ex. 1       25.0   mg                                              Glycofurol 75          100.0  mg                                              Miglyol 812            35.0   mg                                              Cremophor RH 40        90.0   mg                                              Butylhydroxyanisole(BHA)                                                                             0.2    mg                                              Formulation Example D:                                                        Product of Ex. 1       10.0   mg                                              Tetraglycol            10.0   mg                                              Myritol                5.0    mg                                              Cremophor RH 40        75.0   mg                                              alpha-Tocopherol       0.1    mg                                              ______________________________________                                    

The individual components of these formulations, as well as the methodsfor their preparation, are fully described in British Patent Application2 222 770, the contents of which are incorporated herein by reference.

We claim:
 1. A non-immunosuppressive, cyclophilin-binding compound ofFormula II ##STR5## where W' is MeBmt or dihydro-MeBmt;X is αAbu, Val,Thr, Nva or MeOThr; R is Sar or (D)-MeAla; Y is MeLeu, γ-hydroxy-MeLeu,MeIle, MeVal, MeThr, MeAla, MeaIle or MeaThr; and Z is Val, Leu, MeValor MeLeu;provided that 1) when Y is MeLeu or MeAla then Z is MeVal orMeLeu; and 2) when W' is MeBmt, R is Sar, and Y is γ-hydroxy-MeLeu, thenZ is other than Val.
 2. A compound according to claim 1 of Formula II##STR6## in which W' is MeBmt or dihydro-MeBmt;Y is γ-hydroxy-MeLeu,MeVal, MeThr, or MeIle; and Z is Val or MeVal;provided that when W' isMeBmt, Y' is other than γ-hydroxy-MeLeu.
 3. A non-immunosuppressive,cyclophilin-binding compound selected from the group consisting of:dihydro-MeBmt!¹ - γ-hydroxy-MeLeu!⁴ -Ciclosporin, MeVal!⁴ -Ciclosporin,Mele!⁴ -Ciclosporin, MeThr!⁴ -Ciclosporin, Nva!² - γ-hydroxy-MeLeu!⁴-Ciclosporin, γ-hydroxy-MeLeu!⁴ - γ-hydroxy-MeLeu!⁶ -Ciclosporin,MeVal!⁵ -Ciclosporin, and MeOThr!² - (D)MeAla!³ - MeVal!⁵ -Ciclosporin.4. A pharmaceutical composition comprising a compound as described inclaims 1, 2 or 3 together with a pharmaceutically acceptable carrier ordiluent, therefor.
 5. The compound of claim 3 which is MeIle!⁴-ciclosporin.
 6. The pharmaceutical composition according to claim 4comprising MeIle!⁴ -ciclosporin and a pharmaceutically acceptablecarrier therefor.